Impact of Growth Conditions on the Viability of Trichoderma asperellum during Storage.

As excellent biocontrol agents and plant growth promoters, Trichoderma species are agriculturally important. Trichoderma spp. cultures can be produced using solid-state or submerged cultivation, the latter being much less labor intensive and easier to control and automate. The aim of the study was to investigate the ability to increase the shelf-life of T. asperellum cells by optimizing cultivation media and upscaling the submerged cultivation process. Four different cultivation media were used with or without the addition of Tween 80 and stored with or without incorporation into peat, and viability, expressed as CFU/g, was assessed during one year of storage in an industrial warehouse. The addition of Tween 80 had a positive effect on the biomass yield. The culture medium played a major role in the ability of the mycelium to produce spores, which in turn influenced the amount of CFU. This effect was less pronounced when the biomass was mixed with peat prior to storage. A procedure that increases the number of CFU in a peat-based product formulation is recommended, namely, incubation of the mixture at 30 °C for 10 days prior to storage at 15 °C over an extended period of time.

Identification of Saccharomyces cerevisiae genes involved in the resistance to phenolic fermentation inhibitors.

Saccharomyces cerevisiae was exposed to inhibitory concentrations of the three phenolic phenylpropanoids: coniferyl aldehyde, ferulic acid, and isoeugenol. Deoxyribonucleic acid microarray analysis was employed as one approach to generate a set of candidate genes for deletion mutant analysis to determine the potential contribution of the corresponding gene products to the resistance against toxic concentrations of phenolic fermentation inhibitors. Three S. cerevisiae deletion mutants with increased sensitivity to coniferyl aldehyde were identified: yap1Delta, atr1Delta, and flr1Delta. The rate of reduction of coniferyl aldehyde to coniferyl alcohol decreased sixfold when the gene encoding the transcriptional activator Yap1p was deleted, and threefold when the Yap1p-controlled genes encoding Atr1p and Flr1p were deleted. Growth, glucose consumption, and ethanol formation progressed after a lag phase during which coniferyl aldehyde reduction and coniferyl alcohol formation occurred. The results link ATR1, FLR1, and YAP1 by their ability to confer resistance to coniferyl aldehyde and show that deletion of any of these three genes impairs the ability of S. cerevisiae to withstand coniferyl aldehyde and detoxify it by reduction. Furthermore, the results suggest that overexpression of ATR1, FLR1, and YAP1 is of interest for the construction of novel yeast strains with improved resistance against inhibitors in lignocellulose hydrolysates.

Heterologous expression of barley and wheat oxalate oxidase in an E. coli trxB gor double mutant.

Oxalate oxidase catalyses the degradation of oxalic acid to carbon dioxide and hydrogen peroxide and is of commercial importance for clinical analyses of oxalate in biological samples. Novel potential applications for oxalate oxidase include the prevention of the formation of calcium oxalate incrusts in pulp and paper manufacture and rapid determination of oxalic acid in process waters. The potential in using oxalate-degrading enzymes in industrial processes increases the interest in finding systems for heterologous expression. Oxalate oxidase from barley is a secreted multimeric glycosylated manganese-containing enzyme with several disulfide bridges, which have been found to be essential for the catalytic activity. Attempts to achieve expression of active heterologous oxalate oxidase in bacteria have up to now met little success. In this study, one oxalate-oxidase-encoding cDNA from barley and two from wheat were cloned and tested with regard to expression in Escherichia coli. The results suggest that the selection of a novel commercially available E. coli host strain, which has the ability to form disulfide bridges in heterologous proteins expressed in its cytoplasm, was important for successful expression. Although a considerable part of the heterologous protein was produced in an insoluble and inactive form, this strain, E. coli Origami B(DE3), in addition yielded soluble and active barley and wheat oxalate oxidase. One of the wheat cDNAs, Ta(M)OXO1, gave three-fold higher activity than the barley cDNA, Hv(H)OXO1, while the other wheat cDNA, Ta(M)OXO2, gave no detectable activity. This indicates that the choice of cDNA was also critical despite the high identity between the cDNAs and the encoded polypeptides (88-89% on the nucleotide level and 88-92% on the amino-acid level). Gel filtration of cell extracts containing heterologous barley and wheat oxalate oxidase resulted in an increase in the activity. This indicates that low molecular weight inhibitory compounds were present in the E. coli lysates but could be removed by the introduction of a purification step.

Effect of different forms of alkali treatment on specific fermentation inhibitors and on the fermentability of lignocellulose hydrolysates for production of fuel ethanol.

Treatment with alkali, particularly overliming, has been widely used as a method for the detoxification of lignocellulose hydrolysates prior to ethanolic fermentation. However, the mechanisms behind the detoxification effect and the influence of the choice of cation have not been well understood. In this study, a dilute acid hydrolysate of spruce and an inhibitor cocktail consisting of six known inhibitors were used to investigate different alkali detoxification methods. The various treatments included the addition of calcium hydroxide, sodium hydroxide, potassium hydroxide, and ammonia to pH 10.0 and subsequent adjustment of the pH to 5.5 with either sulfuric or hydrochloric acid as well as treatment with the corresponding amounts of calcium, sodium, and potassium as sulfate or chloride salts at pH 5.5. An RP-HPLC method was developed for the separation of 18 different inhibitors in the hydrolysate, including furaldehydes and phenolics. Detection and quantification were carried out by means of UV, DAD, and ESI-MS in negative mode. Treatment of the spruce hydrolysate with alkali resulted in up to approximately 40% decrease in the concentration of furaldehydes. The effects on the aromatic compounds were complex. Furthermore, SFE was performed on the precipitate formed during alkali treatment to evaluate the inhibitor content of the precipitate, and the following RP-HPLC analysis implied that potential inhibitors were removed mainly through conversion rather than through filtration of precipitate. Parallel experiments in which sulfuric acid or hydrochloric acid was used for acidification to pH 5.5 after alkali treatment indicated that the choice of anion did not affect the removal of inhibitors. Detoxification with calcium hydroxide and ammonia resulted in better fermentability using Saccharomyces cerevisiae than detoxification with sodium hydroxide. The results from the experiments with the inhibitor cocktail indicated that the positive effects of alkali treatment are difficult to explain by removal of the inhibitors only and that possible stimulatory effects on the fermenting organism warrant further attention.

Supercritical fluid extraction of a lignocellulosic hydrolysate of spruce for detoxification and to facilitate analysis of inhibitors.

This work describes a novel approach to detoxify lignocellulosic hydrolysates and facilitate the analysis of inhibitory compounds, namely supercritical fluid extraction (SFE). The efficiency of the fermentation of lignocellulosic dilute-acid hydrolysates depends upon the composition of the hydrolysate and the organism used. Furthermore, it has been shown that inhibitors in the hydrolysate reduce the fermentation yield. This knowledge has given rise to the need to identify and remove the inhibiting compounds. Sample clean-up or work-up steps, to provide a clean and concentrated sample for the analytical system, facilitate the characterization of inhibitors, or indeed any compound in the hydrolysates. Removal of inhibitors was performed with countercurrent flow supercritical fluid extraction of liquid hydrolysates. Three different groups of inhibitors (furan derivatives, phenolic compounds, and aliphatic acids) and sugars were subsequently analyzed in the hydrolysate, extracted hydrolysate, and extract. The effect of the SFE treatment was examined with respect to fermentability with Saccharomyces cerevisiae. Not only did the extraction provide a clean and concentrated sample (extract) for analysis, but also a hydrolysate with increased fermentability as well as lower concentrations of inhibitors such as phenolics and furan derivatives.

Treatment with lignin residue: a novel method for detoxification of lignocellulose hydrolysates.

Acid hydrolysis of lignocellulose to hydrolysates intended for production of fuel ethanol results in the formation of byproducts in addition to fermentable sugars. Some of the byproducts, such as phenolic compounds and furan aldehydes, are inhibitory to the fermenting microorganism. Detoxification of the hydrolysates may be necessary for production of ethanol at a satisfactory rate and yield. The lignin residue obtained after hydrolysis is a material with hydrophobic properties that is produced in large amounts as a byproduct within an ethanol production process based on lignocellulosic raw materials. We have explored the possibility of using this lignin residue for detoxification of spruce dilute-acid hydrolysates prior to fermentation with Saccharomyces cerevisiae. Three dilute-acid hydrolysates of spruce were treated with lignin residue, which in all cases resulted in improved fermentability in terms of productivity and yield of ethanol. The effect was improved by washing the lignin before treatment, by using larger amounts of lignin in the treatment, and by performing the treatment at low temperature. Treatment with the lignin residue removed up to 53% of the phenolic compounds and up to 68% of the furan aldehydes in a spruce dilute-acid hydrolysate. A larger fraction of furfural was removed compared to the less hydrophobic 5-hydroxymethylfurfural.